The Hedgehog (Hh) ligands (Shh, Ihh, and Dhh) signal through two membrane receptors (Ptc1 and Ptc2), the regulatory membrane protein Smoothened (SMO), and the Gli transcription factors. Ptc1 represses SMO in the absence of ligand while Hh binding results in Ptc1 inhibition and activation of SMO. Activation of Gli-dependent transcription by Hh-SMO is referred to as the canonical pathway; in contrast, Gli-independent functions of Hh proteins are classified as non-canonical. Gli activation requires the presence of the co-receptors Cdo/Boc and relocalization of SMO to a structure known as the primary cilium. Most human cancers are characterized by hyperactivation of the canonical Hh pathway due to upregulation of Hh ligands, loss-of-function of Ptc1, or gain-of-function of SMO. Remarkably, upregulation of Hh proteins is strictly associated with epithelial cancers. We identified novel signaling components downstream of SMO that lead to Gli activation and uncovered that SMO acts as a classical Gi-coupled receptor that stimulates signals involved in cell cycle regulation, survival, and migration. We have established novel paradigms revealing that Shh can activate small G proteins of the Rho family and lead to cytoskeletal rearrangements and changes in cell motility. We and others have found that Ptc1 induces apoptosis in the absence of Hhs via its C-terminal domain (CTD), and independently of SMO. Based on this evidence, we hypothesize that non-canonical signals, generated directly by Ptc1 or indirectly through SMO, are the main players in Hh-regulation of cell cycle progression, survival and migration. We will: 1) Characterize the mechanistic basis of non-canonical Hh signaling in proliferation, survival, and migration. We will first determine the potential implication of the primary cilium and the co- receptors Cdo/Boc in these responses. We will also investigate the mechanistic aspects of activation of small GTPases by SMO, and their involvement in cell migration. 2) Study the mechanism of regulation of cell cycle progression and apoptosis by Ptc1 independently of SMO/Gli activation. We will first determine whether membrane localization of Ptc1 CTD is necessary to drive cell death, and will identify the minimal fragment of the CTD required for apoptosis. Secondly, we will investigate the mechanism of Ptc1- induced Akt dephosphorylation, and the participation of the mitochondrial apoptotic pathway in Ptc1 CTD- induced apoptosis. Lastly, we will study the mechanisms that lead to cell cycle arrest by Ptc1. 3) Establish the contribution of Ptc1 and SMO signals for proliferation and survival of Hh overexpressing cancer cells. Peptides based on Ptc1 MCTD sequence and cyclopamine will be applied alone and in combination to Hh-overexpressing epithelial cancer cells and we will evaluate their effects on proliferation and survival. Overall, these studies will provide insights into the understanding of non-canonical Hedgehog pathway(s) and will set a mechanistic basis for development of new cancer therapeutics targeting Ptc1 functions. PUBLIC HEALTH RELEVANCE: This proposal is aimed at dissecting the basic signaling mechanisms activated by the Hedgehog signaling pathway that are involved in regulation of cell cycle progression, survival and migration. We propose the novel hypothesis that the Hedgehog receptor Ptc1 regulates cell cycle progression and survival independently of the so-called canonical pathway, which involves Smoothened (SMO) and Gli-dependent transcription of target genes. Understanding the specific contributions of Ptc1 and SMO to signaling pathways implicated in growth, survival and cell cycle regulation in normal and cancer cells, will provide the basis for rationale development of therapeutic applications for human health.